ABSTRACT

A-type lamins are components of the peripheral nuclear lamina but also localize in the nuclear interior in a complex with lamina-associated polypeptide (LAP) 2α. Loss of LAP2α and nucleoplasmic lamins in wild-type cells increases cell proliferation, but in cells expressing progerin (a mutant lamin A that causes Hutchinson–Gilford progeria syndrome), low LAP2α levels result in proliferation defects. Here, the aim was to understand the molecular mechanism governing how relative levels of LAP2α, progerin and nucleoplasmic lamins affect cell proliferation. Cells from progeria patients and inducible progerin-expressing cells expressing low levels of progerin proliferate faster than wild-type or lamin A-expressing control cells, and ectopic expression of LAP2α impairs proliferation. In contrast, cells expressing high levels of progerin and lacking lamins in the nuclear interior proliferate more slowly, and ectopic LAP2α expression enhances proliferation. However, simultaneous expression of LAP2α and wild-type lamin A or an assembly-deficient lamin A mutant restored the nucleoplasmic lamin A pool in these cells and abolished the growth-promoting effect of LAP2α. Our data show that LAP2α promotes or inhibits proliferation of progeria cells depending on the level of A-type lamins in the nuclear interior.

INTRODUCTION

The nuclear lamina in metazoan cells is a proteinaceous meshwork underlying the inner nuclear membrane, which defines the mechanical properties of the nucleus (Osmanagic-Myers et al., 2015) and regulates chromatin organization and gene expression (Gruenbaum and Foisner, 2015; Kind et al., 2015). Lamins and various lamin-binding proteins of the inner nuclear membrane are the major components of the lamina. Based on their biochemical properties, sequence similarities and expression patterns, lamins are classified into A-type and B-type lamins (Gruenbaum and Foisner, 2015). In mammals, B-type lamins (lamin B1 and lamin B2) are encoded by different genes (LMNB1 and LMNB2, respectively) and are expressed in most embryonic and adult cells. A-type lamins are encoded by LMNA, which gives rise to two major isoforms, lamin A and lamin C, primarily expressed at later stages of development and in differentiated cells (Gruenbaum and Foisner, 2015). B-type lamins and lamin A are expressed as prelamins and undergo multiple steps of post-translational processing at their C-terminal CaaX sequence. The first steps are common to B-type lamins and lamin A and include farnesylation of the cysteine residue by farnesyltransferase, followed by cleavage of the aaX tripeptide by FACE1 (also known as ZMPSTE24) or FACE2 (RCE1) and carboxymethylation of the cysteine by isoprenyl-cysteine-carboxymethyltransferase (Rusinol and Sinensky, 2006). B-type lamin processing ends at this step, resulting in farnesylated and carboxymethylated mature B-type lamins, which are tightly associated with the inner nuclear membrane. In contrast, prelamin A is further processed by FACE1, removing 15 C-terminal residues including the farnesylated and carboxymethylated cysteine (Pendás et al., 2002). Therefore, mature lamin A and lamin C, which lacks a CaaX box, are not farnesylated and, in addition to their localization at the lamina, are also found in the nuclear interior (Dechat et al., 2010; Kolb et al., 2011; Moir et al., 2000; Naetar et al., 2017).

Progerin-expressing fibroblasts undergo a period of hyperproliferation before going into cell cycle exit. (A) Mean LAP2α fluorescence intensities of one wild-type (WT 1, p17) and three different HGPS primary human fibroblast cell lines (HGPS 1, p17; HGPS 2, p15; and HGPS 3, p13) were measured in 250 nuclei each and plotted in a histogram (n=3). The percentage of nuclei expressing different levels of LAP2α is shown. (B) Histogram of mean progerin fluorescence intensities measured in 300 nuclei each of HGPS cell lines 1, 2 and 3 at indicated passage numbers (n=3). Dotted lines represent standard error of mean (s.e.m.). (C) Growth curves of WT 1 and HGPS cell lines 1, 2 and 3 over 6 days (n=3). (D) Cells were stained with CFSE for 15 min and grown in complete medium for 5 days before FACS analysis. CFSE staining intensity profiles of WT 1 (blue) and three different HGPS lines (different shades of red) are shown. Green line corresponds to signal after 15 min CFSE pulse and the black profile to the unstained control. (E) WT 1 and three different HGPS lines were grown in medium containing EdU. The percentage of EdU-positive cells was determined after 40 h, followed by immunofluorescence analysis. Mean fluorescence LAP2α intensity of 600 nuclei was measured and plotted against the EdU signal per cell. The percentages of EdU-positive cells in the populations of cells with low LAP2α expression (black rectangle in panel A) can be compared with the percentages in cells with high LAP2α expression. *P<0.05, **P<0.005, ***P<0.0005.

Low LAP2α levels in cells with intermediate progerin levels result in hyperproliferation

To investigate a potential causal link between LAP2α expression levels and proliferation in progerin-expressing cells, we used a tightly controllable hTERT-immortalized skin fibroblast system allowing doxycycline-inducible expression of GFP–progerin or GFP–lamin-A as control (Vidak et al., 2015). Progerin and lamin A levels were detected after one day of doxycycline induction and increased within 6–8 days, as revealed by fluorescence intensity measurements and immunoblotting of cell lysates (Fig. 2A; Fig. S2A). Exogenous GFP–progerin levels were similar to endogenous lamin A levels; those of GFP–lamin-A were up to twofold higher than endogenous lamin A levels in the presence of 0.5 µg ml−1 doxycycline (Fig. S2A). We then assessed cell proliferation following addition of 0.5 µg ml−1 doxycycline for GFP–progerin and 0.1 µg ml−1 doxycycline for GFP–lamin-A cells relative to the respective uninduced controls (Fig. 2B). Importantly, at these doxycycline concentrations, expression levels of GFP–lamin-A and GFP–progerin were comparable (Fig. 2A). Proliferation of cells expressing GFP–lamin-A was slightly reduced compared with uninduced cells; cells expressing GFP–progerin initially (1–4 days induction) proliferated faster than uninduced controls and cells expressing GFP–lamin-A. At days 5–8 post-induction, when progerin levels further increased (Fig. S2A), GFP–progerin fibroblasts exhibited slower proliferation. Hence, in agreement with our observation in HGPS cells (see above) and previous reports (Bridger and Kill, 2004), these data suggest that lower progerin levels correlate with increased proliferation compared with controls, whereas higher progerin expression levels eventually lead to reduced proliferation. Together, these data indicate a progerin-specific growth-promoting phenotype at initial phases of induction.

Loss of LAP2α increases proliferation of WT cells and cells with low progerin expression. (A) Mean GFP-lamin A (GFPLA) and GFP–progerin fluorescence intensities were measured in 250 nuclei each from hTERT-immortalized fibroblasts following doxycycline induction of GFP–progerin (0.5 µg ml−1 doxycycline) and GFP–LA (0.1 µg ml−1 doxycycline) for 2, 3, 4, 5 and 7 days, and plotted in a histogram (n=3). (B) Growth curves of hTERT-immortalized fibroblasts inducibly expressing GFP–LA or GFP–progerin grown in the presence of 0.1 and 0.5 µg ml−1 doxycycline, respectively, for 7 days. The number of cells was normalized to the respective uninduced control (n=3). (C) hTERT-fibroblasts inducibly expressing GFP–LA or GFP–progerin were transfected either with a scrambled control siRNA or siRNA targeting LAP2α (siLAP2α) on two consecutive days prior to doxycycline induction. The cell number was determined after 3 days cultivation in the absence (−Dox) or presence (+Dox) of 1 µg ml−1 doxycycline (n=3). The results are shown as the ratio of cell numbers for siLAP2α versus scrambled siRNA control (n=3). (D) GFP–LA and GFP–progerin expression was induced in hTERT-immortalized fibroblasts by addition of 1 µg ml−1 doxycycline for 24 h. The cells were transfected either with a scrambled control siRNA or siLAP2α on two consecutive days and the cell number determined at post-induction day 6 (n=3). The results are shown as the ratio for siLAP2α versus scrambled siRNA control (n=3). (E) Immunoblot analysis of total cell lysates using anti-LAP2α and anti-actin (loading control) antibodies. *P<0.05, **P<0.005, ***P<0.0005.

To test whether the initial hyperproliferation of progerin-expressing cells is causally linked to reduced LAP2α levels, as previously proposed (Chojnowski et al., 2015), we analyzed the effect of siRNA-mediated downregulation of LAPα at different stages of doxycycline induction. Human TERT-fibroblasts were transfected with a siSCRAMBLE control oligonucleotide or with siRNA oligonucleotide targeting LAP2α, which caused downregulation of LAP2α protein by ∼90% (Fig. 2E). In uninduced cells and cells expressing GFP–progerin or GFP–lamin-A after 3 days of doxycycline induction, downregulation of LAPα resulted in increased proliferation (Fig. 2C), as previously reported for WT cells (Naetar et al., 2008). Thus, under these conditions (intermediate progerin expression and low levels of LAP2α), progerin-expressing cells behave like WT cells in proliferation assays. However, after 6 days of ectopic protein induction, progerin-expressing cells proliferated more slowly and no longer showed hyperproliferation upon LAP2α downregulation, whereas proliferation of lamin A-expressing control cells remained unchanged (Fig. 2D). Thus, the proliferation response of cells to LAP2α downregulation differs for cells with low or high levels of progerin expression.

Effect of ectopic LAP2α expression on proliferation of patient cells depends on the levels of nucleoplasmic lamins. (A) Human primary fibroblasts were transfected either with a control GFP-expressing construct (GFP ctrl) or a human myc-LAP2α-expressing construct (myc-LAP2α) on two consecutive days and counted on days 4–8 (n=3). (B) Primary human fibroblasts were fixed with 4% paraformaldehyde and processed for immunofluorescence using anti-lamin A specific antibody. Scale bars: 10 µm. (C) Fluorescence intensity of the lamin A signal was measured across nuclei (dotted line in B) and plotted. *P<0.05, **P<0.005.

To elucidate why LAP2α expression rescues cell proliferation in late passage HGPS cells, but slows down proliferation in mid-passage cells, we tested the level of nucleoplasmic lamin A, previously shown to be downregulated in progeria cells (Vidak et al., 2015). Immunofluorescence microscopy using a lamin A specific antibody (not detecting progerin) revealed that nucleoplasmic lamins are still present in mid-passage progeria cells (HGPS 1 at p17 and HGPS 2 at p15), but absent in late passage HGPS 2 (p21) cells (Fig. 3B,C; Fig. S3). These data suggest that the effect of LAP2α on the proliferation of progerin-expressing cells depends on the level of nucleoplasmic lamins, whereby high nucleoplasmic lamin levels result in decreased proliferation and low nucleoplasmic lamin levels result in a proliferation-promoting effect of ectopic LAP2α.

Similar effects were observed in the controllable GFP–progerin-expressing hTERT-fibroblast line. After 6 and 8 days, expression of LAP2α in doxycycline-induced progerin-expressing cells, which have high progerin levels (Fig. S2A) and low levels of nucleoplasmic lamins (Fig. 4C), significantly enhanced cell proliferation to the level of cells expressing GFP–lamin-A (6 days) or by ∼50% compared with the control at 8 days (Fig. 4A,B; Fig. S4A). To rescue the nucleoplasmic lamin A pool, we expressed either myc-tagged WT lamin A or a myc-tagged mutant ΔK32 lamin A. The latter is a lamin variant linked to a severe form of congenital muscular dystrophy (CMD) in humans (Quijano-Roy et al., 2008). In LmnaΔK32/ΔK32 knock-in mice, ΔK32 lamin A failed to assemble into the lamina and mislocalized to the nucleoplasm (Bertrand et al., 2012; Pilat et al., 2013). Using a lentiviral transduction system, we achieved a transfection efficiency of up to 90% in hTERT-immortalized fibroblasts; myc-tagged proteins were readily detectable by immunoblot analysis (Fig. S4C,D). Immunofluorescence analysis using an anti myc-tag antibody revealed that WT lamin A localized mostly at the nuclear periphery, but a small fraction was also detected in the nuclear interior, whereas the laminAΔK32 mutant was present predominantly in the nuclear interior, significantly enriching the nucleoplasmic lamin pool (Fig. S4B). For cell proliferation analyses, cells expressing GFP–progerin were transduced with control constructs encoding WT lamin A, laminAΔK32, LAP2α or GFP, and with a combination of constructs encoding lamin A plus LAP2α or laminAΔK32 plus LAP2α on two consecutive days prior to the induction of GFP–progerin. Analyses were carried out up to 8 days post-induction (Fig. 4A,B). The nucleoplasmic pool of lamin A was partially restored by ectopic lamin A expression (Fig. 4D,F, arrowheads) and significantly enriched upon expression of laminAΔK32 (Fig. 4E,G, arrowheads). Fibroblasts transfected with a GFP control plasmid showed slower proliferation 4–6 days after progerin induction (Fig. 4A,B), whereas ectopic expression of LAP2α enhanced proliferation of the progerin-expressing hTERT-fibroblasts (Fig. 4A,B). This increase in cell proliferation correlated with loss of nucleoplasmic lamins, as observed by measuring the ratio of nucleoplasmic lamin A staining to rim staining in 100 cells (Fig. 4C). Interestingly, ectopic expression of either WT lamin A or laminAΔK32 mutant alone also led to an increase in proliferation compared with progerin-expressing control cells (Fig. 4A,B). Strikingly, however, ectopic expression of LAP2α together with either WT lamin A or laminAΔK32 (rescuing the nucleoplasmic lamin A pool) almost completely inhibited the proliferation-promoting effect of LAP2α (Fig. 4A,B). Together, our data demonstrate that the effect of LAP2α expression on proliferation in progeria cells is highly dependent on the level of nucleoplasmic A-type lamins.

Re-expression of LAP2α in the presence of nucleoplasmic lamins does not rescue proliferation of progerin-expressing cells. (A) hTERT-immortalized fibroblasts inducibly expressing GFP–progerin were transfected either with a single construct (control GFP-expressing plasmid, human myc-lamin A- or human myc-LAP2α-expressing construct) or with a combination of myc–lamin A- and myc-LAP2α-expressing constructs on two consecutive days prior to the induction of progerin expression and counted on days 2–8 post-induction (n=3). *P<0.05 comparing cells expressing myc-LAP2α with cells expressing myc-lamin A+LAP2α. (B) The same as for A, except that myc–laminAΔK32 expressing construct was used instead of WT lamin A-expressing construct (n=3). *P<0.05 comparing cells expressing myc-LAP2α with cells expressing myc–lamin AΔK32+LAP2α. (C) Ratios of nucleoplasmic to peripheral mean lamin A fluorescence intensities were calculated from 100 GFP–progerin-expressing nuclei in immunofluorescence images and plotted in a histogram (***P<0.0005). (D) Immunofluorescence analysis of cells transfected with human myc-lamin A using anti-myc (red) and lamin A-specific antibody (greyscale). DAPI (blue) was used to stain DNA. (E) Immunofluorescence analysis of cells transfected with human myc-lamin AΔK32 using anti-myc (red) and lamin A-specific antibody (greyscale). DAPI (blue) was used to stain DNA. Circles in D and E indicate cells not expressing myc-lamin A or myc-lamin AΔK32 and arrowheads point to the cells represented in the intensity profiles in F and G. (F,G) Mean fluorescence intensities of the lamin A signal were measured across nuclei (dotted lines in D and E) and plotted. Scale bars: 20 µm.

DISCUSSION

In this study, we analyzed the proliferation phenotype in progerin-expressing and WT cells in the absence and presence of LAP2α and its relation to the levels of progerin and nucleoplasmic A-type lamins. The HGPS cell lines tested not only showed highly heterogeneous expression levels of LAP2α and progerin within cells in the culture, as previously reported (Vidak et al., 2015), but they also showed heterogeneous proliferation rates. HGPS cell lines expressing intermediate levels of progerin proliferated faster than the respective control cell line, potentially linked to a cell population in the culture with low LAP2α levels. In contrast, HGPS cell cultures expressing high levels of progerin exhibited slower proliferation. Because this variability in different HGPS cell lines could be partially rooted in the different genetic background of patients from whom the cell lines were derived, we also analyzed hTERT-expressing fibroblasts that can be cultured under tightly controllable conditions. Using this cell system, we observed a similar initial hyperproliferation following induction of progerin expression compared with lamin A expression, and a subsequent rapid decline in the number of proliferative cells at later stages, when progerin levels were high.

Intriguingly, a significant fraction of HGPS cells with low LAP2α expression levels still proliferated, indicating that the loss of LAP2α in HGPS cells does not immediately lead to proliferation defects as previously reported (Chojnowski et al., 2015; Vidak et al., 2015). Indeed, these proliferating and LAP2α-negative cells in HGPS cultures could account for the overall hyperproliferation of the cultures, based on previous observations showing that loss of LAP2α in WT fibroblasts increases proliferation (Dorner et al., 2006; Naetar et al., 2008). Thus, LAP2α might have a dual role in the development of HGPS, depending on progerin levels and disease progression. In cells with low progerin levels, reduced levels of LAP2α could cause an initial period of hyperproliferation, whereas at later stages, when progerin expression increases, low LAP2α levels correlate with decreased proliferation. Accordingly, overexpression of LAP2α in cells expressing low progerin levels impairs proliferation, whereas it rescues proliferation in cells expressing high progerin levels. However, how LAP2α is downregulated in a subset of proliferating cells in growing HGPS cultures remains elusive.

Why does loss or gain of LAP2α have different effects in cells with low or high expression of progerin? Immunofluorescence analysis revealed that low progerin-expressing cells, such as progeria lines derived from young patients, contained significant levels of lamin A in the nuclear interior, whereas the nucleoplasmic pool of A-type lamins was greatly reduced in progeria cells derived from an older patient that express higher levels of progerin. In addition, prolonged cultivation of cells derived from young patients, which is known to increase progerin levels (Vidak et al., 2015), significantly decreases the nucleoplasmic pool of lamins. Is it indeed the presence or absence of lamin A/C in the nuclear interior that defines whether LAP2α has proliferation-promoting or proliferation-inhibiting functions? To address this question, we expressed LAP2α alone or in combination with ectopic lamin A in cells expressing GFP–progerin that had lost lamin A/C in the nuclear interior. Expression of LAP2α, which does not rescue the nucleoplasmic pool of lamin A/C in these cells, increased cell proliferation (Vidak et al., 2015). In contrast, ectopic expression of LAP2α together with ectopic WT lamin A or an assembly-deficient disease-linked laminAΔK32 mutant rescued the nucleoplasmic pool of lamin A to different extents but, importantly, did not enhance cell proliferation. Thus, LAP2α primarily has a growth-inhibiting effect in cells containing lamins in the nuclear interior, but a growth-promoting effect in cells that lack nucleoplasmic lamins.

How are these different functions of LAP2α mediated? LAP2α and A-type lamins were found to interact with the cell cycle regulatory protein pRb (Markiewicz et al., 2002), which represses E2F-dependent transcription to mediate cell cycle arrest (Kaelin, 1999). In addition, LAP2α interacts with E2F/pRb target genes and represses an E2F-dependent reporter gene dependent on the presence of pRb (Dorner et al., 2006). The observation that the expression of WT LAP2α in Lap2α−/− cells, but not the expression of a LAP2α truncation mutant lacking its pRb and lamin A interaction domains, reduced cell proliferation (Naetar et al., 2008) suggests that LAP2α can inhibit proliferation only in collaboration with or in a complex with nucleoplasmic lamin A and pRb (Dorner et al., 2006; Naetar et al., 2008; Pilat et al., 2013). Thus, LAP2α has a proliferation-inhibiting effect only in cells containing lamin A/C in the nuclear interior. This effect is probably mediated at the G1–S phase transition of the cell cycle (Dorner et al., 2006).

Upon progerin accumulation in prolonged cell culture, the nucleoplasmic pool of A-type lamins is lost, probably by forming heteromeric complexes with progerin at the nuclear periphery. This renders LAP2α no longer capable of inhibiting cell proliferation via pRb and its proliferation-promoting function becomes evident. Potential mechanistic insight into its growth-promoting effect are provided by our recent finding that LAP2α interacts with euchromatic regions in the mammalian genome and regulates lamin-A–chromatin association (Gesson et al., 2016). Contrary to previous reports showing preferential binding of lamin A to heterochromatic regions termed lamina-associated domains (LADs) (Kind et al., 2015), we showed that lamin A also binds to euchromatic regions outside of LADs largely overlapping with LAP2α-associated genomic regions in the nucleoplasm. Loss of LAP2α caused a reorganization of lamin A–chromatin association, leading to changes in epigenetic profiles and gene expression (Gesson et al., 2016). Thus, loss of nucleoplasmic lamin A in progeria cells can change epigenetic pathways and gene expression. In line with this hypothesis, we found decreased expression of extracellular matrix genes in progeria versus WT cells and rescue of ECM gene expression upon ectopic expression of LAP2α (Vidak et al., 2015). Although the ECM has previously been shown to affect cell proliferation of progeria cells (de la Rosa et al., 2013; Hernandez et al., 2010; Vidak et al., 2015), the molecular pathways are not completely understood, particularly regarding if and which cell cycle phases are affected.

Together, our studies show that LAP2α has at least two different functions, a growth-inhibiting activity requiring the presence of lamin A/C in the nuclear interior and a growth-promoting role in the absence of nucleoplasmic lamin A/C.

Proliferation assays

For growth curves, hTERT-TetOn-Pro cell lines or primary human fibroblasts were plated in triplicates on six-well plates at a density of 40,000 cells per well and grown for 7 days under the indicated conditions (0.1–1.0 µg ml−1). For the siRNA experiments, cells were plated at a density of 100,000 cells per well and grown for 3 days. Cell numbers were determined using a Casy cell counter model TTC (Schärfe System). EdU incorporation assays were performed using the EdU–Click imaging kit according to the manufacturer's manual (Base Click). Positive cells were quantified using a LSM 710 confocal microscope (Carl Zeiss) and 25×0.8 NA oil immersion objective.

The CFSE staining assay was performed using Vybrant® CFDA SE Cell Tracer Kit (Molecular Probes, V12883). A stock solution of 10 mM CFDA SE (carboxyfluorescein diacetate succinimidyl ester; CFSE) was prepared freshly by dissolving the contents of one vial (component A) in 90 µl high-quality DMSO (component B) and diluting in PBS to 20 µM. Cultures (at 40% confluence) of primary human fibroblasts on a 6 cm plate were incubated with 20 µM CFSE probe in pre-warmed (37°C) PBS for 15 min at 37°C. Cultures were grown in fresh complete medium for an additional 5 days at 37°C, washed once with PBS, trypsinized, collected in a 15 ml Falcon tube and pelleted by centrifugation at 1100 rpm for 5 min. The cell pellet was resuspended in 1 ml of PBS and the CFSE signal detected by flow cytometry using the BD FACS Calibur multicolor flow cytometer (BD Biosciences).

Statistical analysis

All calculations were performed using Microsoft Excel and Graphpad Prism software. Experimental data are reported as means of a minimum of three biological replicates. The two-tailed Student's t-test was used for statistical analyses. Error bars represent standard deviation (s.d.), except in the growth curves where error bars represent the standard error of the mean (s.e.m.). Statistical significance was classified as follows: *P<0.05, **P<0.005 and ***P<0.0005.

Acknowledgements

We thank Maciej Szafraniec for help with the cloning of lentiviral constructs. We are grateful to R. Goldman and S. A. Adam (Northwestern University, Chicago, IL) and to I. Yudushkin, D. Blaas and E. Ogris (MFPL Vienna, Austria) for generous gifts of reagents.

R.F. was funded by the Austrian Science Fund (FWF grant P26492-B20), T.D. was funded by a Herzfelder'sche Familienstiftung and Progeria Research Foundation (Innovator Award, PRF2011-37) and S.V. was supported by a doctoral program funded by the Austrian Science Fund (FWF, DK W1220). Deposited in PMC for immediate release.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

(2011). Lamin A and lamin C form homodimers and coexist in higher complex forms both in the nucleoplasmic fraction and in the lamina of cultured human cells. Nucleus2, 425-433.doi:10.4161/nucl.2.5.17765

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